1. Field of the Invention
The present invention relates generally to an image reader, and more particularly to an image reader that is usable in an autoradiographic system employing an X-ray film or storable phosphor sheet, a detection system using an electron microscope, a radiation diffraction image detection system, and a fluorescence detection system, the image reader being equipped with photoelectric conversion means having a photoelectron multiplication function, a current-voltage conversion circuit, and a logarithmic conversion circuit.
2. Description of the Related Art
In autoradiography, a radioactive labeling substance is injected into a living organism, and the living organism or part of the tissue of the living organism is sampled. This sample is stacked on a photosensitive material such as an X-ray film for a fixed time and is exposed to light. Based on the exposed part of the photosensitive material, the positional information on the radioactive labeling substance in the sample is obtained.
This autoradiography has been widely utilized in the following manner. For example, a radioactive labeling substance is injected into a medium which contains an organism-oriented high molecular substance such as the tissue of a living organism, protein, a nucleic acid, etc. The high molecular substance with the radioactive labeling substance, the derivative, or the decomposed substance, is separated on a gel support body by a separation operation such as gel electrophoresis. The gel support body is stacked on a radiation film such as a high-sensitivity X-ray film for a fixed time and is exposed to light. Based on the positional information on the radiation labeling substance obtained from the exposed part of the radiation film, the separation and identification of the high molecular substance, or the molecular weight measurement and characteristic evaluation of the high molecular substance, is performed. This autoradiography has also been utilized effectively in determining the base (nucleotide) sequence of a nucleic acid such as deoxyribonucleic acid (DNA).
In addition, as shown in Japanese Patent Publication Nos. 1(1989)-60784, 1(1989)-60782, and 4(1992)-3952 and Japanese Unexamined Patent Publication No. 10(1998)-3134, autoradiography employing a storable phosphor sheet instead of a conventional radiation film has also been put to practical use. The storable phosphor sheet is used as a photosensitive material for obtaining the positional information on a radioactive labeling substance. The storable phosphor sheet has a stimulatable phosphor layer, which contains a stimulatable phosphor. The stimulatable phosphor absorbs and stores energy of radiation when irradiated with the radiation, and emits photostimulated luminescent light, having a quantity of light corresponding to the quantity of the stored radiation energy, when excited with an electromagnetic wave such as visible light, infrared light, etc.
In autoradiography employing the storable phosphor sheet, a sample containing a radioactive labeling substance is stacked on the storable phosphor sheet for a fixed time, and the storable phosphor sheet absorbs at least part of radiation energy emitted from the radioactive labeling substance. Then, the radiation energy is emitted as photostimulated luminescent light from the storable phosphor sheet by scanning the storable phosphor sheet with an electromagnetic wave such as laser light, etc. The emitted, photostimulated luminescent light is photoelectrically detected, and the positional information on the radioactive labeling substance in the sample is obtained.
Japanese Unexamined Patent Publication Nos. 59(1984)-15843, 61(1986)-51738, and 61(1986)-93538, for example, disclose a detection system, which uses an electron microscope, for irradiating electron beams to a living organism and detecting the image of the living organism, and a radiation diffraction image detection system for irradiating radiation to a sample, detecting a radiation diffraction image, and making a structural analysis of the sample. As shown in these publications, a stimulatable phosphor is employed as a material for detecting electron beams or radiation. The stimulatable phosphor has the property of absorbing and storing the energy of electron beams or radiation when irradiated with the electron beams or radiation and also emitting photostimulated luminescent light having a quantity of light corresponding to the quantity of the stored electron-beam or radiation energy when excited with an electromagnetic wave in a specific wavelength region. Electron beams are irradiated to a metal or non-metal sample, and the diffraction image or transmitted image of the sample is detected. Based on the detected image, an element analysis, a composition analysis of the sample, a structural analysis of the sample, etc., are made.
There is also a fluorescence detection system using a fluorescent dye (labeling substance) instead of the radioactive labeling substance used in the autoradiographic system. This detection system can make an evaluation of a gene sequence and a gene expression level, an evaluation of the excretion, absorption, metabolism path, and state of an injected substance in a laboratory mouse, a separation and identification of protein, a measurement of a molecular weight, and an evaluation of characteristics, by reading a fluorescence image. For example, after a fluorescent dye is added into a solution containing a plurality of DNA fragments that are electrophoresed, the DNA fragments are electrophoresed on a gel support body. Alternatively, a plurality of DNA fragments are electrophoresed on a gel support body containing a fluorescent dye. Or, after a plurality of DNA fragments are electrophoresed on a gel support body, the gel support body is immersed into a solution containing a fluorescent dye. Next, after the thus-electrophoresed DNA fragments have been labeled, the fluorescent dye is excited with excitation light and fluorescent light is emitted. The emitted fluorescent light is detected, and an image is generated to detect DNA distribution on the gel support body. Alternatively, after a plurality of DNA fragments are electrophoresed on a gel support body, they are denatured. Next, at least some of the denatured DNA fragments are transferred onto a transfer support body such as nitrocellulose by Southern blotting, and target DNA and complementary DNA (or RNA) are labeled with a fluorescent dye to prepare a DNA or RNA probe. The DNA or RNA probe and the denatured DNA fragments are hybridized so that only the DNA or RNA probe and cDNA fragments are selectively labeled. The fluorescent light, emitted by exciting the fluorescent dye with excitation light, is detected to generate an image. A distribution of target DNAs on the transfer support body can also be detected. Furthermore, a cDNA probe, complementary to DNA containing a target gene labeled with a labeling substance, is prepared and hybridized with DNA on the transfer support body. After it is bound with the labeled cDNA, enzyme is brought into contacted with a fluorescent substrate. The fluorescent substrate is turned into a fluorescent substance which emits fluorescent light. The fluorescent light, emitted by exciting the generated fluorescent substance with excitation light, is detected and an image is generated. With this image, a distribution of target DNAs on the transfer support body can also be detected. These fluorescence detection systems have the advantage that they can easily detect a gene sequence, etc., without using a radioactive substance.
As described in the xe2x80x9cgene expression analysis employing a microarray chip (Experimental Medical Series, Vol. 17, Jan. 1999, pp. 61 to 65)xe2x80x9d and xe2x80x9cgene medical science (Vol. 4, No. 1, 2000 (Medical Do)),xe2x80x9d attention has recently been paid to application of the aforementioned fluorescence detection system to the gene expression analysis technique employing a test piece such as a microarray chip, a macroarray chip, a DNA chip, etc.
The gene expression analysis technique employs a test piece, in which a wide variety of known different biomolecules (such as cDNA, oligo-DNA, other DNAs, PNA, EST, etc.) having already been translated are disposed and spotted in matrix form as probes (detecting substances) on the surface of a substrate, such as a membrane filter, a glass substrate, a glass slide substrate, a silicon substrate, etc., at predetermined intervals of a few hundreds xcexcm or less (e.g., 20 to 200 xcexcm) by a spotter, etc. The test piece is called a microarray chip, a macroarray chip, a DNA chip, etc., depending on the substrate type, the substrate size, the number of spots, the spot size, the probe type, the target type, etc.
On the other hand, biomolecules such as cDNA, genomic DNA, RNA (messenger RNA, etc.), dNTP, PNA, etc., are labeled with a radioactive isotope or fluorescent dye and prepared as targets that are detected.
When an analysis is made by the aforementioned gene express analysis technique, DNA (an example of an organism-oriented substance) taken out from the cell of a healthy person (target A) labeled with a fluorescent dye a, and DNA taken out from a target B with a genetic disease labeled with a fluorescent dye b, are dropped on a microarray chip with a pipette so that each DNA is hybridized with cDNA on the microarray chip. Then, each cDNA on the microarray chip is scanned with excitation light that separately excites the fluorescent dyes a and b, and fluorescent light is photoelectrically detected for each cDNA. From the results of detection corresponding to the positions on the microarray chip from which fluorescent light was emitted, it is decided which of the cDNAs has been hybridized with each DNA. Finally, DNAs expressed or lost due to the genetic disease are specified by comparing the cDNAs hybridized between the two targets A and B.
The autoradiographic system employing an X-ray film or storable phosphor sheet, the detection system using an electro microscope, the radiation diffraction image detection system, and the fluorescence detection system (hereinafter referred to as various detection systems), incidentally, are equipped with an image reader. In the image reader, an image carrier, which carries a fluorescent image or radiation image related to a living organism, such as a storable phosphor sheet, a gel support body, or a transfer support body carrying an image, is scanned with excitation light. Fluorescent light or photostimulated luminescent light, emitted from the image carrier, is detected photoelectrically by photoelectric conversion means, and an image is generated.
The photoelectric conversion means, for photoelectrically detecting fluorescent light or photostimulated luminescent light, employs an avalanche photodiode (APD), a photomultiplier, etc., which have a photoelectron multiplication function, because the fluorescent light or photostimulated luminescent light is feeble light whose intensity (light quantity) is not so strong.
In the current-voltage conversion circuit for converting the electrical signal, output from the photoelectric conversion means, to a voltage signal (hereinafter also referred to as I-V conversion), it is common practice from the point of view of simplicity of construction and cost to perform a detection by DC coupling, employing resistance.
It is also standard practice, in consideration of enlargement of a dynamic range and a manner of being visually observed when a result of detection is imaged, to logarithmically compress an I/V-converted voltage signal with a logarithmic conversion circuit.
The size of a pixel that is read is required to be about 200 xcexcm or less, and in connection with the reading speed, the sampling frequency fS employed in converting the logarithmically-compressed analog voltage signal to a digital voltage signal (hereinafter also referred to as A/D conversion) is of an intermediate speed of about 10 to 1000 KHz.
That is, each detection circuit in the aforementioned various detection systems is constructed so that it performs I/V conversion employing resistance, logarithmic conversion, and intermediate-speed sampling.
On the other hand, presently there is a strong demand for information of higher resolution. The intensity (light quantity) of fluorescent light or photostimulated luminescent light that is incident on the aforementioned photoelectric conversion means has been increasingly reduced. In other words, a region that is detected has been shifted to a high-sensitivity side.
However, when feeble light is irradiated, for example, to a photomultiplier, photons incident on the photoelectric surface of the photomultiplier become sparse with respect to time. For this reason, a current which is observed when electrons generated by the photons are multiplied and reach the collector becomes pulsed. A voltage signal which is obtained by performing I/V conversion on the current output from the photomultiplier, using a conversion resistance of about 1000 kxcexa9, also becomes pulsed.
Even when light is feeble, stable direct-current (DC) output is obtained in a region where the light intensity is great to some degree. However, output pulses that are approximately even in height are continuously connected, as the light intensity becomes smaller. That is, the DC output fluctuates. If the light intensity becomes even smaller, output pulses that are approximately even in height will be dispersed, and the frequency of pulses will change, depending on light intensity. As a result, a single pulse region is obtained. That is, if the light intensity becomes smaller, the DC output will go to a state close to photon counting.
In this case, if a voltage signal is obtained by a conventional I/V conversion circuit employing resistance and is converted to a digital signal, a non-signal portion in a pulsed signal not synchronized with a sampling clock signal will sometimes be sampled during A/D conversion, as shown in FIG. 13. As a result, there are voltage signals that are not sampled. That is, voltage signals are sampled in a manner having considerably great level variations, and the S/N ratio of an image signal is degraded.
To avoid this, there is a method of providing a filter before A/D conversion. However, if a logarithmically compressed signal is passed through the filter and converted to a digital signal, the transient response characteristic (nonlinear response characteristic) portion in the logarithmic conversion circuit will be sampled and therefore the signal will vary widely and signal linearity will be lost. In addition, it will become difficult to discriminate background noise from signals. As a result, a very coarse image is obtained and there arises a problem that quantification will become difficult.
Furthermore, if an analog filter is provided at the input side in order to avoid repeated noise, which corresponds to a relatively intermediate speed sampling frequency fS of about 10 to 1000 KHz, during A/D conversion, ripple or tailing will occur when a pulsed signal is input to the A/D converter circuit and will have adverse effects on an image. Practically, there are also circumstances where A/D conversion must be performed without antialiasing.
The present invention has been made in view of the circumstances mentioned above. Accordingly, it is an object of the present invention to provide an image reader that can be used in the aforementioned various detection systems. Another object of the invention is to provide an image reader which is capable of generating a satisfactory image by obtaining a suitable image signal even in the case of detecting light that is more feeble than conventional light. Still another object of the invention is to provide an image reader in which quantification is easy even in the case of detecting light that is more feeble than conventional light.
To achieve the objects mentioned above, there is provided an image reader comprising:
excitation light irradiation means for irradiating excitation light to an image carrier carrying a fluorescent image or radiation image related to a living organism;
photoelectric conversion means, which has a photon multiplication function, for photoelectrically detecting feeble light emitted by irradiation of the excitation light, and outputting a current signal which has a magnitude corresponding to the light quantity of the feeble light;
a current-voltage conversion circuit for converting the current signal to a voltage signal; and
a logarithmic conversion circuit for logarithmically converting the voltage signal converted by the current-voltage conversion circuit;
wherein an integration circuit, DC-coupled with the photoelectric conversion means, is employed as the current-voltage conversion circuit.
The expression xe2x80x9cimage carrier carrying a fluorescent image or radiation image related to a living organismxe2x80x9d refers to an image carrier carrying a fluorescent image or radiation image related to a living organism, such as a storable phosphor sheet, a gel support body, or a transfer support body carrying an image which is read in the autoradiographic system employing an X-ray film or storable phosphor sheet, the detection system using an electron microscope, the radiation diffraction image detection system, or the fluorescence detection system.
More specifically, in the case of the autoradiographic system employing a storable phosphor sheet, the image carrier refers to a storable phosphor sheet obtained in the following manner. That is, a radioactive labeling substance is injected into a medium that contains an organism-oriented high molecular substance such as the tissue of a living organism, protein, a nucleic acid, etc. The high molecular substance with the radioactive labeling substance is stacked on a storable phosphor sheet for a fixed time and is exposed to light. The storable phosphor sheet carries an image represented by the radioactive labeling substance. Also, in the case of the fluorescence detection system using a fluorescent dye as a labeling substance, the image carrier refers to a gel support body, a microarray chip, a macroarray chip, or a DNA chip, subjected to a hybridization process, which carries an image represented by a fluorescent dye.
In the case of a system for detecting fluorescence (self-fluorescence) emitted from a living organism or a dye contained in the tissue piece, the living organism or tissue piece itself may be used as the image carrier.
In the image reader of the present invention, it is desirable that an analog-digital conversion circuit be provided after the logarithmic conversion circuit. The analog-digital conversion circuit is used for performing analog-digital conversion on the logarithmically converted voltage signal at a sampling frequency of between 10 KHz and 1 MHz.
In the image reader of the present invention, it is desirable that the current-voltage conversion circuit be constructed so that it can stop integral action of the integration circuit.
It is desirable that the integration circuit of the image reader of the present invention have a differential amplifier, an integration capacitor connected between one input terminal and an output terminal of the differential amplifier, and a reset field-effect transistor, connected in parallel with the integration capacitor, for resetting the integration capacitor.
The integration circuit in this form is grouped into two types of construction: a mirror integration construction in which an integration capacitor is connected between an inverting input terminal and an output terminal of a differential amplifier; and a boot strap integration construction in which an integration capacitor is connected between a non-inverting input terminal and an output terminal of a differential amplifier. In the present invention, the mirror integration construction is preferable to the boot strap integration construction, because an offset compensation circuit described later becomes structurally simpler.
The expression xe2x80x9cbetween one input terminal and an output terminal of the differential amplifierxe2x80x9d varies depending on the integration circuit construction. For instance, in the case of mirror integration construction, it refers to xe2x80x9cbetween an inverting input terminal and an output terminal.xe2x80x9d In the case of boot strap integration construction, it refers to xe2x80x9cbetween a non-inverting input terminal and an output terminal.xe2x80x9d Note that the integration capacitor may be connected directly to the output terminal of the differential amplifier, or it may be connected to the output terminal through resistance.
In the case where the integration circuit in this form is used, noise that occurs during setting or resetting of the integration circuit causes charge injection, which results in offset voltage (DC component) that is output from the integration circuit. This offset voltage is the direct cause of a variation in the output voltage of the integration circuit, because it can vary according to the capacitance of an integration capacitor or capacitance of a reset field-effect transistor (device variations).
Therefore, the image reader of the present invention further comprises an offset compensation circuit which has a compensation field-effect transistor. The compensation field-effect transistor has a drain terminal and a source terminal, connected between the other input terminal of the differential amplifier and a reference potential, and also has a gate terminal to which control voltage is input for adjusting a capacitance variation in the integration capacitor and/or a capacitance variation in the reset field-effect transistor.
In this case, it is preferable that a compensation capacitor having the same capacitance as the capacitance of the integration capacitor be connected in parallel with the compensation field-effect transistor.
The expression xe2x80x9ca drain terminal and a source terminal, connected between the other input terminal of the differential amplifier and a reference potentialxe2x80x9d means that one of the drain and source terminals is connected to the aforementioned other input terminal, while the other of the drain and source terminals is connected to the reference potential.
The expression xe2x80x9cbetween the other input terminal of the differential amplifier and a reference potentialxe2x80x9d varies in integration circuit construction. For example, in the case of mirror integration construction, it refers to xe2x80x9cbetween a non-inverting input terminal and a ground potential (reference potential).xe2x80x9d In the case of boot strap integration construction, it refers to xe2x80x9cbetween an inverting input terminal and an operating-point setting potential (reference potential).xe2x80x9d
In the integration circuit of the image reader of the present invention, it is preferable that the reset field-effect transistor and the compensation filed-effect transistor have small input capacitance and feedback capacitance. For instance, they may be junction field-effect transistors.
The image reader of the present invention may further comprise means for cooling the photoelectric conversion means. It is preferable that the cooling means be constructed, for example, of a Peltier element.
The application of the aforementioned offset compensation circuit is not limited to an image reader which is used in an autoradiographic system employing an X-ray film or storable phosphor sheet, a detection system using an electron microscope, a radiation diffraction image detection system, and a fluorescence detection system. The offset compensation circuit is also applicable to a voltage-signal acquisition circuit, which comprises: (1) an integration circuit having a differential amplifier, an integration capacitor connected between one input terminal and an output terminal of the differential amplifier, and a reset field-effect transistor, connected in parallel with the integration capacitor, for resetting the integration capacitor; and (2) a logarithmic conversion circuit for logarithmically converting a voltage signal output from the integration circuit. As with the aforementioned case, it is preferable that the reset field-effect transistor and the compensation filed-effect transistor employ junction field-effect transistors.
According to the image reader of the present invention, an integration circuit, DC-coupled with photoelectric conversion means, is employed as a current-voltage conversion circuit that is provided before a logarithmic conversion circuit. Therefore, even in the case of detecting such extremely feeble light that a single pulse signal is input as a current signal, pulse signals are integrated and converted to a voltage signal. As a result, an image signal which is more suitable than a conventional one can be obtained.
That is, there is no need to input a pulse waveform, as it is, to an A/D conversion circuit or logarithmic conversion circuit. Therefore, even if an input current signal is a current signal in the form of a pulse, a non-signal portion in the pulse waveform not synchronized with a sampling clock signal can be prevented from being sampled, except when the pulse current signal coincides with reset timing. As a result, the S/N ratio of an image can be enhanced compared with the prior art.
Since the integration circuit is provided before the logarithmic conversion circuit, the transient response characteristic portion of the logarithmic conversion circuit can be prevented from being sampled. As a result, there is no problem of signal linearity being lost or quantification becoming difficult.
The image reader of the present invention employs the integration circuit, which has a differential amplifier, an integration capacitor connected between one input terminal and an output terminal of the differential amplifier, and a reset field-effect transistor, connected in parallel with the integration capacitor, for resetting the integration capacitor. Particularly, if the integration circuit has a mirror integration construction in which an integration capacitor is connected between the inverting input terminal and an output terminal of a differential amplifier, an integration circuit with a relatively intermediate response speed corresponding to a sampling frequency of 10 KHz to 1 MHz for A/D conversion can be made structurally simple.
In the case of employing the aforementioned integration circuit, there is provided an offset compensation circuit having a compensation field-effect transistor. The compensation field-effect transistor has a drain terminal and a source terminal, connected between the other input terminal of the differential amplifier and a reference potential, and also has a gate terminal to which a control voltage is input for adjusting a capacitance variation in the integration capacitor and/or a capacitance variation in the reset field-effect transistor. As a result, the offset voltage that may occur because of charge injection during integral action can be reduced.